Cébron Aurélie, Norini Marie-Paule, Beguiristain Thierry, Leyval Corinne
Laboratoire des Interactions Microorganismes-Minéraux-Matière Organique dans les Sols UMR7137, Nancy Université, CNRS, Faculté des Sciences, B.P. 239, 54506 Vandoeuvre-les-Nancy Cedex, France.
J Microbiol Methods. 2008 May;73(2):148-59. doi: 10.1016/j.mimet.2008.01.009. Epub 2008 Feb 2.
Real-Time PCR based assays were developed to quantify Gram positive (GP) and Gram negative (GN) bacterial populations that are capable of degrading the polycyclic aromatic hydrocarbons (PAH) in soil and sediment samples with contrasting contamination levels. These specific and sensitive Real-Time PCR assays were based on the quantification of the copy number of the gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDalpha), involved in the initial step of the aerobic metabolism of PAH. The PAH-RHDalpha-GP primer set was designed against the different allele types present in the data base (narAa, phdA/pdoA2, nidA/pdoA1, nidA3/fadA1) common to the Gram positive PAH degraders such as Rhodococcus, Mycobacterium, Nocardioides and Terrabacter strains. The PAH-RHDalpha-GN primer set was designed against the genes (nahAc, nahA3, nagAc, ndoB, ndoC2, pahAc, pahA3, phnAc, phnA1, bphAc, bphA1, dntAc and arhA1) common to the Gram negative PAH degraders such as Pseudomonas, Ralstonia, Commamonas, Burkholderia, Sphingomonas, Alcaligenes, Polaromonas strains. The PCR clones for DNA extracted from soil and sediment samples using the designed primers showed 100% relatedness to the PAH-RHDalpha genes targeted. Deduced from highly sensitive Real-Time PCR quantification, the ratio of PAH-RHDalpha gene relative to the 16S rRNA gene copy number showed that the PAH-bacterial degraders could represent up to 1% of the total bacterial community in the PAH-contaminated sites. This ratio highlighted a positive correlation between the PAH-bacterial biodegradation potential and the PAH-contamination level in the environmental samples studied.
基于实时荧光定量PCR的检测方法被开发出来，用于量化革兰氏阳性（GP）和革兰氏阴性（GN）细菌群体，这些细菌能够降解土壤和沉积物样本中污染程度不同的多环芳烃（PAH）。这些特异且灵敏的实时荧光定量PCR检测方法基于对编码PAH环羟基化双加氧酶（PAH-RHDα）α亚基的基因拷贝数进行定量，该酶参与PAH有氧代谢的初始步骤。PAH-RHDα-GP引物组是针对革兰氏阳性PAH降解菌（如红球菌属、分枝杆菌属、类诺卡氏菌属和地杆菌属菌株）数据库中存在的不同等位基因类型（narAa、phdA/pdoA2、nidA/pdoA1、nidA3/fadA1）设计的。PAH-RHDα-GN引物组是针对革兰氏阴性PAH降解菌（如假单胞菌属、罗尔斯通氏菌属、丛毛单胞菌属、伯克霍尔德氏菌属、鞘氨醇单胞菌属、产碱菌属、极地单胞菌属菌株）中常见的基因（nahAc、nahA3、nagAc、ndoB、ndoC2、pahAc、pahA3、phnAc、phnA1、bphAc、bphA1、dntAc和arhA1）设计的。使用设计引物从土壤和沉积物样本中提取的DNA进行PCR克隆，结果显示与目标PAH-RHDα基因的相关性为100%。从高度灵敏的实时荧光定量PCR定量结果推断，PAH-RHDα基因与16S rRNA基因拷贝数的比率表明，PAH细菌降解菌在PAH污染位点的细菌群落中所占比例可达1%。该比率突出了所研究环境样本中PAH细菌生物降解潜力与PAH污染水平之间的正相关关系。
